title: Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.
abstract: Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, Graduate University for Advanced Studies, SOKENDAI, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Muto
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ohkura
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kotani
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Higashijima
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001522:Behavior, Animal / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism*; D009043:Motor Activity / Q000502:physiology; D009046:Motor Neurons / Q000166:cytology / Q000502:physiology*; D009119:Muscle Contraction / Q000502:physiology; D013116:Spinal Cord / Q000166:cytology*; D015027:Zebrafish / Q000033:anatomy & histology* / Q000235:genetics / Q000502:physiology*
chemical_list: D007202:Indicators and Reagents; D049452:Green Fluorescent Proteins; D002118:Calcium